The invention relates to certain anti-p185-HER-2/neu antibodies and the use of said antibodies in assays for the determination of HER-2/neu expression levels in biological samples. The antibodies of the invention are accurate indicators of HER-2/neu overexpression in human cancerous tissue using immunocytochemical or immunohistochemical assays. These reagents are useful for identifying cancer patients who have the greatest probability of relapse and/or the least likelihood of survival and, as a result, may be likely to benefit from adjuvant therapy.
Prognostic factors often help predict relapse and survival in patients suffering from cancer. The presence of certain factors that are indicative of a greater probability of relapse and/or a low probability of survival may suggest that adjuvant therapy is appropriate. High-dose chemotherapy or autologous bone marrow transplantation are possible treatment regimens after surgery. The existence of reliable prognostic factors to predict relapse and survival are important since aggressive cancer therapy is costly and is frequently accompanied by toxic side effects. Likewise, the absence of such factors may indicate that less intensive therapy is required. Prognostic factors that have been used to predict relapse in breast cancer patients include tumor size (Carter et al. Cancer 63, 181-187 (1989)), number of lymph nodes involved (Carter et al., supra), histologic grade (Henson et al. Cancer 68, 2142-2149 (1991)), and the presence of estrogen or progesterone receptors (Osborne, in Breast Diseases, Harris, J. R. et al., eds. 2nd ed. J. B. Lippincott, pp. 301-325 (1991)). Recently, a variety of molecular markers have shown potential as prognostic factors for identifying cancer patients, particularly those suffering from breast and ovarian cancer, that are most likely to benefit from aggressive cancer therapy. Molecular markers that may be important include measurements of DNA content, cell proliferation and oncogene expression. Oncogene expression has received some attention by investigators in view of the apparent correlation between expression of the HER-2/neu oncogene and poor prognosis in patients with breast cancer. However, as explained below, this correlation has not been observed by all investigators or has been observed in only a subset of patients examined.
The HER-2/neu oncogene encodes a membrane-associated glycoprotein referred to as p185HER-2/neu having extracellular, transmembrane and intracellular domains, with the extracellular domain having homology to that of the epidermal growth factor receptor. The human gene, designated as c-erbB-2, HER-2, or neu , was reported by Semba et al. (Proc. Natl. Acad. Sci. USA 82, 6497-6501 (1985)); Coussens et al. (Science 230, 1132-1139 (1985)) and King et al. (Science 229, 974-976 (1985)). A related rat gene was reported by Schecter et al (Science 229, 976-978 (1985)).
Increased expression of the HER-2/neu oncogene in tumor cells and cell lines has been reported by several groups (Coussens et al., supra; King et al., supra). The increased expression of HER-2/neu results from gene amplification or increased expression of the single copy gene. These observations suggested that HER-2/neu may be overexpressed in human cancer tissue. Slamon and colleagues (Slamon et al. Science 235, 177-182 (1987); Slamon et al. Science 244, 707-712 (1989)) examined HER-2/neu expression levels in tumors taken from a large sample of breast and ovarian cancer patients. It was found that nearly 30% of those patients had amplification of the HER-2/neu gene, that the amplification was associated with overexpression, and that overexpression of HER-2/neu was associated with a poor clinical outcome (increased relapse and low survival rate) particularly in node-positive breast cancer patients. The correlations reported by Slamon have been confirmed in a number of studies (see, for example, Ro et al. Cancer Res. 49, 6941-6944 (1989); Walker et al. Brit. J. Cancer 60, 426-429 (1989); Wright et al. Cancer Res. 49, 2087-2090 (1989); Berchuck et al. Cancer Res 50, 4087-4091 (1990); Kallioniemi et al. Int. J. Cancer 49, 650-655 (1991); Rilke et al. Int. J. Cancer 49, 44-49 (1991)). However, other investigators have not found a significant correlation between prognosis and HER-2/neu overexpression in breast and ovarian cancer (see, for example, Van de Vijver et al. N. Engl. J. Med. 319, 1239-1245 (1988); Zhou et al. Oncogene 4, 105-108 (1989); Clark et al. Cancer Res. 51, 944-948 (1991); Kury et al. Eur. J. Cancer 26, 946-949 (1990); Rubin et al. Am. J. Obstet. Gynecol. 168, 162-169 (1993)). Presently, it is not clear in the art as to the reliability of HER-2/neu overexpression as a prognostic factor in breast and other cancers.
Most studies reported to date that have examined HER-2/neu expression levels in human breast cancer tissue specimens have employed immunohistochemical analysis of fixed paraffin-embedded tissue samples. A variety of anti-p185HER-2/neu antibodies have been generated and used in evaluating HER-2/neu expression (see, for example, van de Vijver et al. Cancer Cells 7, 385-391 (1989); Gullick et al. Int. J. Cancer 40, 246-254 (1987); Corbett et al. J. Path. 161, 15-25 (1990); Fendly et al. Cancer Res. 50, 1550-1558 (1990); Slamon et al. Cancer Cells 7, 371-380 (1989)). In view of the contradictory conclusions regarding the predictive value of HER-2/neu expression levels in breast cancer tissue, Press et al. (Cancer Res. 54, 2771-2777 (1994)) undertook a systematic evaluation of 28 different anti-p185HER-2/neu antibodies using multi-tumor tissue blocks. They observed significant variability in the detection of HER-2/neu expression levels in the same tissue samples by different antibodies. It has become apparent that the antibody used in the analysis of HER-2/neu expression is a crucial reagent that can significantly affect the reliability of HER-2/neu expression as a prognostic tool.
U.S. Pat. No. 4,968,603 discloses methods for screening patients suffering from breast and ovarian cancer for HER-2/neu expression or amplification. Expression of the HER-2/neu gene can be measured in one instance by immunohistochemical staining using an antibody raised against part or all of the HER-2/neu polypeptide. This disclosure does not provide anti-p185HER-2/neu antibodies nor does it suggest the variability with which different anti-p185HER-2/neu antibodies may react with p185HER-2/neu protein in biological samples. PCT Application No. WO89/10412 discloses antibodies to HER-2/neu protein generated by using NIH 3T3 cells transfected with a HER-2/neu full-length cDNA clone as the immunogen. Also disclosed are methods for detecting HER-2/neu overexpression using anti-p185HER-2/neu antibodies. PCT Application No. WO89/06692 discloses antibodies raised to NIH 3T3 cells transfected with full-length HER-2/neu cDNA clone and discloses methods for detecting tumors expressing HER-2/neu using anti-p185HER-2/neu antibodies. PCT Application No. WO93/03741 discloses antibodies raised to SK-BR-3 human breast cancer cells as an immunogen. None of these applications describe the reaction of anti-p185HER-2/neu antibodies with human cancer tissue samples. In addition, none of these applications address the problem of variable reactivity of anti-p185HER-2/neu antibodies with HER-2/neu protein in tissue samples.
It has been recently reported (Muss et al. N. Engl. J. Med. 330, 1260-1266 (1994)) that node-positive breast cancer patients treated with high-dose chemotherapy had significantly longer time to relapse and longer survival time if their tumors had HER-2/neu overexpression, while patients with little or no HER-2/neu expression showed no significant benefit to increased dosage.
In view of the potential importance of HER-2/neu overexpression in predicting response to treatment in certain cancers, it is desirable to identify reagents which will accurately and reliably measure levels of HER-2/neu expression. In particular, it is desirable to develop anti-p185HER-2/neu antibodies which are useful for detection of HER-2/neu expression in cell and tissue specimens using immunostaining techniques. It is desirable that the antibodies react strongly with biological samples that exhibit HER-2/neu overexpression while, at the same time, react poorly or not at all with samples expressing HER-2/neu at normal levels.
The invention relates to anti-p185HER-2/neu monoclonal antibodies or antibody fragments thereof which bind to denatured epitopes comprising a subset of amino acids residues 96-191 of the HER-2/neu protein as shown in FIG. 1B (SEQ ID NO: 3). The antibodies recognize HER-2/neu protein in biological samples that have been treated with a fixative, but recognize poorly, or not at all, HER-2/neu protein in its native conformation in biological samples.
Anti-p185HER-2/neu antibodies of the invention will react strongly with biological sample having HER-2/neu overexpression, but will react weakly or not at all with samples having normal levels of HER-2/neu expression. Preferably, the antibodies will react strongly with at least 80% of cancer specimens (tissues or cells) which overexpress HER-2/neu and will react weakly or not at all with cancer specimens which express HER-2/neu at normal levels. In one embodiment, the antibody is selected from the group consisting of antibodies 9C2 and 11G5. Also encompassed by the invention are hybridoma cell lines producing such antibodies.
The invention also relates to a method for detecting HER-2/neu expression in a biological sample by contacting the sample with an anti-p185HER-2/neu antibody of the present invention under conditions appropriate for antibody binding to the sample, and determining the extent of antibody binding to the sample. Preferably, the biological sample is a cell or tissue specimen derived from stomach, lung, breast, pancreatic, prostate or ovarian cancer which is treated with a fixative prior to analysis.
The invention also relates to a kit for use in detecting HER-2/neu expression in a biological sample using the antibodies of the present invention.